Methods for the identification of inhibitors of homocitrate synthase as antibiotics

ABSTRACT

The present inventors have discovered that homocitrate synthase is essential for fungal pathogenicity. Specifically, the inhibition of homocitrate synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, homocitrate synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit homocitrate synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

[0001] The invention relates generally to methods for the identificationof antibiotics, preferably antifungals that affect the biosynthesis oflysine. This application is co-pending with our application entitled“Methods for the Identification of Inhibitors of α-AminoadipateReductase as Antibiotics”.

BACKGROUND OF THE INVENTION

[0002] Filamentous fungi are the causal agents responsible for manyserious pathogenic infections of plants and animals. Since fungi areeukaryotes, and thus more similar to their host organisms than, forexample bacteria, the treatment of infections by fungi poses specialrisks and challenges not encountered with other types of infections. Onesuch fungus is Magnaporthe grisea, the fungus that causes rice blastdisease. It is an organism that poses a significant threat to foodsupplies worldwide. Other examples of plant pathogens of economicimportance include the pathogens in the genera Agaricus, Alternaria,Anisogramma, Anthracoidea, Antrodia, Apiognomonia, Apiosporina,Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera,Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora,Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria,Chrysomyxa, Cladosporium, Claviceps, Cochliobolus, Coleosporium,Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium,Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora,Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia,Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,Endocronartium, Endothia, Entyloma, Epichloe, Erysiphe, Exobasidium,Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma,Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella,Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium,Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus,Irpex, Kabatiella, Kabatina, Laetiporus, Laetisaria, Lasiodiplodia,Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina,Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina,Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium,Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella,Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma,Penicillium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus,Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis,Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera,Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium,Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina,Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia,Typhula, Uncinula, Urocystis, Uromyces, Ustilago, Valsa, Venturia,Verticillium, Xylaria, and others. Related organisms in theclassification, oomycetes, that include the genera Albugo, Aphanomyces,Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara,Pseudoperonospora, Pythium, Sclerophthora, and others are alsosignificant plant pathogens and are sometimes classified along with thetrue fungi. Human diseases that are caused by filamentous fungi includelife-threatening lung and disseminated diseases, often a result ofinfections by Aspergillus fumigatus. Other fungal diseases in animalsare caused by fungi in the genera, Fusarium, Blastomyces, Microsporum,Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,Cryptococcus, other Aspergilli, and others. The control of fungaldiseases in plants and animals is usually mediated by chemicals thatinhibit the growth, proliferation, and/or pathogenicity of the fungalorganisms. To date, there are less than twenty known modes-of-action forplant protection fungicides and human antifungal compounds.

[0003] A pathogenic organism has been defined as an organism thatcauses, or is capable of causing disease. Pathogenic organisms propagateon or in tissues and may obtain nutrients and other essential materialsfrom their hosts. A substantial amount of work concerning filamentousfungal pathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et. al. (1999)Microb Pathog 27: 123-31 (PMID: 10455003)) have shown that the deletionof either of two suspected pathogenicity related genes encoding analkaline protease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encoding,aspergillopepsin (PEP) in Aspergillus fumigatus, had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990 a, b, c, d, and e) U.S. Pat.Nos. 4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck &Co. Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) MolMicrobiol 36:1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

[0004] There are also a number of papers that report less clear results,showing neither full pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the are A transcriptional activator on the pathogenicity ofAspergillus fumigatus. Tang et al. (Tang, C. M., J. M. Smith, et al.(1994) Infect Immun 62: 5255-60 (PMID: 7960102)) using the relatedfungus, Aspergillus nidulans, observed that a mutation inpara-aminobenzoic acid synthesis prevented mortality in mice, while amutation in lysine biosynthesis had no significant effect on themortality of the infected mice.

[0005] Therefore, it is not currently possible to determine whichspecific growth materials may be readily obtained by a pathogen from itshost, and which materials may not. Surprising, especially in light ofthe results showing that a lysine biosynthesis mutation in thefilamentous fungus, Aspergillus nidulans, had no significant effect onthe pathogenicity in a mouse model system (Tang, C. M., J. M. Smith, etal. (1994) Infect Immun 62: 5255-60 (PMID: 7960102)), we have found thatMagnaporthe grisea that cannot synthesize their own lysine are entirelynon-pathogenic on their host organism. To date there do not appear to beany publications demonstrating an anti-pathogenic effect of theknock-out, over-expression, antisense expression, or inhibition of thegenes or gene products involved in lysine biosynthesis in filamentousfungi. Thus, it has not been shown that the de novo biosynthesis oflysine is essential for fungal pathogenicity. Our co-pending applicationentitled “Methods for the Identification of Inhibitors of α-AminoadipateReductase as Antibiotics” shows that the disruption of lysinebiosynthesis as the result of a disruption of the gene encoding theenzyme activity, alpha-aminoadipate-semialdehyde dehydrogenase, alsoresults in a non-pathogenic phenotype for M. grisea. Thus, it would bedesirable to determine the utility of the enzymes involved in lysinebiosynthesis for evaluating antibiotic compounds, especially fungicides.If a fungal biochemical pathway or specific gene product in that pathwayis shown to be required for fungal pathogenicity, various formats of invitro and in vivo screening assays may be put in place to discoverclasses of chemical compounds that react with the validated target gene,gene product, or biochemical pathway, and are thus candidates forantifungal, biocide, and biostatic materials.

SUMMARY OF THE INVENTION

[0006] Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding homocitrate synthase in Magnaporthegrisea prevents or inhibits the pathogenicity of the fungus. Thus, thepresent inventors have discovered that homocitrate synthase is essentialfor normal rice blast pathogenicity, and can be used as a target for theidentification of antibiotics, preferably fungicides. Accordingly, thepresent invention provides methods for the identification of compoundsthat inhibit homocitrate synthase expression or activity. The methods ofthe invention are useful for the identification of antibiotics,preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows the reaction performed by the homocitrate synthase(HCS1) reaction. The Substrates/Products are AcetylCoA+H₂O+2-oxoglutarate and the Products/Substrates are2-hydroxybutane-1,2,4-tricarboxylate+CoA. The function of thehomocitrate synthase enzyme is the interconversion of Acetyl CoA,2-oxoglutarate, and H₂O to 2-hydroxybutane-1,2,4-tricarboxylate and CoA.This reaction is part of the lysine biosynthesis pathway.

[0008]FIG. 2 shows a digital image showing the effect of HCS1 genedisruption on Magnaporthe grisea pathogenicity using whole plantinfection assays. Rice variety C039 was inoculated with wild-type andthe transposon insertion strains, KO1-1 and KO1-2. Leaf segments wereimaged at five days post-inoculation.

[0009]FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-1 andKO1-2, were grown in (A) minimal media and (B) minimal media with theaddition of L-lysine, respectively. The x-axis shows time in days andthe y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (—♦—), transposon strainKO1-1 (—▪—), and transposon strain KO1-2 (—▴—).

DETAILED DESCRIPTION OF THE INVENTION

[0010] Unless otherwise indicated, the following terms are intended tohave the following meanings in interpreting the present invention.

[0011] The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

[0012] As used herein, the term “allele” refers to any of thealternative forms of a gene that may occur at a given locus.

[0013] The term “antibiotic” refers to any substance or compound thatwhen contacted with a living cell, organism, virus, or other entitycapable of replication, results in a reduction of growth, viability, orpathogenicity of that entity.

[0014] The term “binding” refers to a non-covalent or a covalentinteraction, preferably non-covalent, that holds two molecules together.For example, two such molecules could be an enzyme and an inhibitor ofthat enzyme. Non-covalent interactions include hydrogen bonding, ionicinteractions among charged groups, van der Waals interactions andhydrophobic interactions among nonpolar groups. One or more of theseinteractions can mediate the binding of two molecules to each other.

[0015] The term “biochemical pathway” or “pathway” refers to a connectedseries of biochemical reactions normally occurring in a cell, or morebroadly a cellular event such as cellular division or DNA replication.Typically, the steps in such a biochemical pathway act in a coordinatedfashion to produce a specific product or products or to produce someother particular biochemical action. Such a biochemical pathway requiresthe expression product of a gene if the absence of that expressionproduct either directly or indirectly prevents the completion of one ormore steps in that pathway, thereby preventing or significantly reducingthe production of one or more normal products or effects of thatpathway. Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

[0016] As used herein, the term “cDNA” means complementarydeoxyribonucleic acid.

[0017] As used herein, the term “CoA” means coenzyme A.

[0018] As used herein, the term “conditional lethal” refers to amutation permitting growth and/or survival only under special growth orenvironmental conditions.

[0019] As used herein, the term “cosmid” refers to a hybrid vector, usedin gene cloning, that includes a cos site (from the lambdabacteriophage). It also contains drug resistance marker genes and otherplasmid genes. Cosmids are especially suitable for cloning large genesor multigene fragments.

[0020] As used herein, the term “dominant allele” refers to a dominantmutant allele in which a discernable mutant phenotype can be detectedwhen this mutation is present in an organism that also contains a wildtype (non-mutant), recessive allele, or other dominant allele.

[0021] As used herein, the term “DNA” means deoxyribonucleic acid.

[0022] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay. “Fungi” (singular: fungus) refers to whole fungi,fungal organs and tissues (e.g., asci, hyphae, pseudohyphae, rhizoid,sclerotia, sterigmata, spores, sporodochia, sporangia, synnemata,conidia, ascostroma, cleistothecia, mycelia, perithecia, basidia and thelike), spores, fungal cells and the progeny thereof. Fungi are a groupof organisms (about 50,000 known species), including, but not limitedto, mushrooms, mildews, moulds, yeasts, etc., comprising the kingdomFungi. They can either exist as single cells or make up a multicellularbody called a mycelium, which consists of filaments known as hyphae.Most fungal cells are multinucleate and have cell walls, composedchiefly of chitin. Fungi exist primarily in damp situations on land and,because of the absence of chlorophyll and thus the inability tomanufacture their own food by photosynthesis, are either parasites onother organisms or saprotrophs feeding on dead organic matter. Theprincipal criteria used in classification are the nature of the sporesproduced and the presence or absence of cross walls within the hyphae.Fungi are distributed worldwide in terrestrial, freshwater, and marinehabitats. Some live in the soil. Many pathogenic fungi cause disease inanimals and man or in plants, while some saprotrophs are destructive totimber, textiles, and other materials. Some fungi form associations withother organisms, most notably with algae to form lichens.

[0023] As used herein, the term “fungicide”, “antifungal”, or“antimycotic” refers to an antibiotic substance or compound that killsor suppresses the growth, viability, or pathogenicity of at least onefungus, fungal cell, fungal tissue or spore.

[0024] In the context of this disclosure, “gene” should be understood torefer to a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides.) The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

[0025] As used in this disclosure, the terms “growth” or “cell growth”of an organism refers to an increase in mass, density, or number ofcells of said organism. Some common methods for the measurement ofgrowth include the determination of the optical density of a cellsuspension, the counting of the number of cells in a fixed volume, thecounting of the number of cells by measurement of cell division, themeasurement of cellular mass or cellular volume, and the like.

[0026] As used in this disclosure, the term “growth conditionalphenotype” indicates that a fungal strain having such a phenotypeexhibits a significantly greater difference in growth rates in responseto a change in one or more of the culture parameters than an otherwisesimilar strain not having a growth conditional phenotype. Typically, agrowth conditional phenotype is described with respect to a singlegrowth culture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

[0027] As used herein, the term “H₂O” means water.

[0028] As used herein, the term “HCS1 ” means a gene encodinghomocitrate synthase activity, referring to an enzyme that catalyses theinterconversion of acetyl-CoA, H₂O, and 2-oxoglutarate with2-hydroxybutane-1,2,4-tricarboxylate and CoA.

[0029] As used herein, the term “heterologous HCS1 gene” means a gene,not derived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea homocitrate synthase, preferably 25%, 50%, 75%, 90%,95%, 99% and each integer unit of activity from 10-100% in ascendingorder.

[0030] As used herein, the term “homocitrate synthase (EC 4.1.3.21) orhomocitrate synthase polypeptide” is synonymous with “the HCS1 geneproduct” and refers to an enzyme that catalyses the interconversion ofacetyl-CoA, H₂O, and 2-oxoglutarate with2-hydroxybutane-1,2,4-tricarboxylate and CoA.

[0031] As used herein, the term “His-Tag” refers to an encodedpolypeptide consisting of multiple consecutive histidine amino acids.

[0032] As used herein, the term “HPLC” means high pressure liquidchromatography.

[0033] As used herein, the terms “hph”, “hygromycin Bphosphotransferase”, and “hygromycin resistance gene” refer to the E.coli hygromycin phosphotransferase gene or gene product.

[0034] As used herein, the term “hygromycin B” refers to anaminoglycosidic antibiotic, used for selection and maintenance ofeukaryotic cells containing the E. coli hygromycin resistance gene.

[0035] “Hypersensitive” refers to a phenotype in which cells are moresensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0036] “Hyposensitive” refers to a phenotype in which cells are lesssensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0037] As used herein, the term “imperfect state” refers to aclassification of a fungal organism having no demonstrable sexual lifestage.

[0038] The term “inhibitor”, as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity of homocitratesynthase or substantially reduces the level of enzymatic activity,wherein “substantially” means a reduction at least as great as thestandard deviation for a measurement, preferably a reduction by 50%,more preferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0039] A polynucleotide may be “introduced” into a fungal cell by anymeans known to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

[0040] As used herein, the term “knockout” or “gene disruption” refersto the creation of organisms carrying a null mutation (a mutation inwhich there is no active gene product), a partial null mutation ormutations, or an alteration or alterations in gene regulation byinterrupting a DNA sequence through insertion of a foreign piece of DNA.Usually the foreign DNA encodes a selectable marker.

[0041] As used herein, the term “LB agar” means Luria's Broth agar.

[0042] The term “method of screening” means that the method is suitable,and is typically used, for testing for a particular property or effectin a large number of compounds. Typically, more than one compound istested simultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “method ofscreening” also refers to determining a set of different properties oreffects of one compound simultaneously.

[0043] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0044] As used herein, the term “mutant form” of a gene refers to a genewhich has been altered, either naturally or artificially, changing thebase sequence of the gene. The change in the base sequence may be ofseveral different types, including changes of one or more bases fordifferent bases, deletions, and/or insertions, such as by a transposon.By contrast, a normal form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

[0045] As used herein, the term “Ni” refers to nickel.

[0046] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0047] As used herein, the term “one form” of a gene is synonymous withthe term “gene”, and a “different form” of a gene refers to a gene thathas greater than 49% sequence identity and less than 100% sequenceidentity with said first form.

[0048] As used herein, the term “pathogenicity” refers to a capabilityof causing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

[0049] As used herein, the term “PCR” means polymerase chain reaction.

[0050] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool; (Altschul, S.F., W.Gish, et al. (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at theNational Center for Biotechnology or using Smith Waterman Alignment(Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7 (PMID:7265238)) as incorporated into GeneMatcher Plus™. It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

[0051] By “polypeptide” is meant a chain of at least two amino acidsjoined by peptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

[0052] As used herein, the term “proliferation” is synonymous to theterm “growth”.

[0053] As used herein, the term “reverse transcriptase-PCR” meansreverse transcription-polymerase chain reaction.

[0054] As used herein, the term “RNA” means ribonucleic acid.

[0055] As used herein, “semi-permissive conditions” are conditions inwhich the relevant culture parameter for a particular growth conditionalphenotype is intermediate between permissive conditions andnon-permissive conditions. Consequently, in semi-permissive conditionsan organism having a growth conditional phenotype will exhibit growthrates intermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially functionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

[0056] “Sensitivity phenotype” refers to a phenotype that exhibitseither hypersensitivity or hyposensitivity.

[0057] The term “specific binding” refers to an interaction betweenhomocitrate synthase and a molecule or compound, wherein the interactionis dependent upon the primary amino acid sequence and/or theconformation of homocitrate synthase.

[0058] As used herein, the term “TLC” means thin layer chromatography.

[0059] “Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

[0060] For the purposes of the invention, “transgenic” refers to anycell, spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

[0061] As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/JUS00/07317, and U.S. 09/658,859. As used herein, the term“transposition” refers to a complex genetic rearrangement processinvolving the movement or copying of a polynucleotide (transposon) fromone location and insertion into another, often within or between agenome or genomes, or DNA constructs such as plasmids, bacmids, andcosmids.

[0062] As used herein, the term “transposon” (also known as a“transposable element”, “transposable genetic element”, “mobileelement”, or “jumping gene”) refers to a mobile DNA element such asthose, for example, described in WO 00/55346, PCT/US00/07317, and U.S.09/658,859. Transposons can disrupt gene expression or cause deletionsand inversions, and hence affect both the genotype and phenotype of theorganisms concerned. The mobility of transposable elements has long beenused in genetic manipulation, to introduce genes or other informationinto the genome of certain model systems.

[0063] As used herein, the term “Tween 20” means sorbitanmono-9-octadecenoate poly(oxy-1,1-ethanediyl).

[0064] As used in this disclosure, the term “viability” of an organismrefers to the ability of an organism to demonstrate growth underconditions appropriate for said organism, or to demonstrate an activecellular function. Some examples of active cellular functions includerespiration as measured by gas evolution, secretion of proteins and/orother compounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

[0065] The present inventors have discovered that disruption of the HCS1gene and/or gene product inhibits the pathogenicity of Magnaporthegrisea. Thus, the inventors are the first to demonstrate thathomocitrate synthase is a target for antibiotics, preferablyantifungals.

[0066] Accordingly, the invention provides methods for identifyingcompounds that inhibit HCS1 gene expression or biological activity ofits gene product(s). Such methods include ligand binding assays, assaysfor enzyme activity, cell-based assays, and assays for HCS1 geneexpression. Any compound that is a ligand for homocitrate synthase mayhave antibiotic activity. For the purposes of the invention, “ligand”refers to a molecule that will bind to a site on a polypeptide. Thecompounds identified by the methods of the invention are useful asantibiotics.

[0067] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0068] a) contacting a homocitrate synthase polypeptide with said testcompound; and

[0069] b) detecting the presence or absence of binding between said testcompound and said homocitrate synthase polypeptide;

[0070] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0071] The homocitrate synthase protein may have the amino acid sequenceof a naturally occurring homocitrate synthase found in a fungus, animal,plant, or microorganism, or may have an amino acid sequence derived froma naturally occurring sequence. Preferably the homocitrate synthase is afungal homocitrate synthase. The cDNA (SEQ ID NO: 1) encoding thehomocitrate synthase protein, the genomic DNA (SEQ ID NO: 2) encodingthe protein, and the polypeptide (SEQ ID NO: 3) can be found herein.

[0072] By “fungal homocitrate synthase” is meant an enzyme that can befound in at least one fungus, and which catalyzes the interconversion ofacetyl-CoA and H₂O and 2-oxoglutarate with2-hydroxybutane-1,2,4-tricarboxylate and CoA. The homocitrate synthasemay be from any of the fungi, including ascomycota, zygomycota,basidiomycota, chytridiomycota, and lichens.

[0073] In one embodiment, the homocitrate synthase is a Magnaporthehomocitrate synthase. Magnaporthe species include, but are not limitedto, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea andMagnaporthepoae and the imperfect states of Magnaporthe in the genusPyricularia. Preferably, the Magnaporthe homocitrate synthase is fromMagnaporthe grisea.

[0074] In various embodiments, the homocitrate synthase can be fromPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

[0075] Fragments of a homocitrate synthase polypeptide may be used inthe methods of the invention, preferably if the fragments include anintact or nearly intact epitope that occurs on the biologically activewildtype homocitrate synthase. The fragments comprise at least 10consecutive amino acids of a homocitrate synthase. Preferably, thefragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430 or at least 440 consecutive amino acidsresidues of a homocitrate synthase. In one embodiment, the fragment isfrom a Magnaporthe homocitrate synthase. Preferably, the fragmentcontains an amino acid sequence conserved among fungal homocitratesynthases.

[0076] Polypeptides having at least 50% sequence identity with a fungalhomocitrate synthase are also useful in the methods of the invention.Preferably, the sequence identity is at least 60%, more preferably thesequence identity is at least 70%, most preferably the sequence identityis at least 80% or 90 or 95 or 99%, or any integer from 60-100% sequenceidentity in ascending order.

[0077] In addition, it is preferred that the polypeptide has at least10% of the activity of a fungal homocitrate synthase. More preferably,the polypeptide has at least 25%, at least 50%, at least 75% or at least90% of the activity of a fungal homocitrate synthase. Most preferably,the polypeptide has at least 10%, at least 25%, at least 50%, at least75% or at least 90% of the activity of the M. grisea homocitratesynthase protein.

[0078] Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

[0079] a) contacting said test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal homocitrate synthase, a polypeptidehaving at least 50% sequence identity with a fungal homocitratesynthase, and a polypeptide having at least 10% of the activity thereof;and

[0080] b) detecting the presence and/or absence of binding between saidtest compound and said polypeptide;

[0081] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0082] Any technique for detecting the binding of a ligand to its targetmay be used in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a homocitrate synthase protein or a fragment or variant thereof,the unbound protein is removed and the bound homocitrate synthase isdetected. In a preferred embodiment, bound homocitrate synthase isdetected using a labeled binding partner, such as a labeled antibody. Ina variation of this assay, homocitrate synthase is labeled prior tocontacting the immobilized candidate ligands. Preferred labels includefluorescent or radioactive moieties. Preferred detection methods includefluorescence correlation spectroscopy (FCS) and FCS-related confocalnanofluorimetric methods.

[0083] Once a compound is identified as a candidate for an antibiotic,it can be tested for the ability to inhibit homocitrate synthaseenzymatic activity. The compounds can be tested using either in vitro orcell based assays. Alternatively, a compound can be tested by applyingit directly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

[0084] By decrease in growth, is meant that the antifungal candidatecauses at least a 10% decrease in the growth of the fungus or fungalcells, as compared to the growth of the fungus or fungal cells in theabsence of the antifungal candidate. By a decrease in viability is meantthat at least 20% of the fungal cells, or portion of the funguscontacted with the antifungal candidate are nonviable. Preferably, thegrowth or viability will be decreased by at least 40%. More preferably,the growth or viability will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal growth and cellviability are known to those skilled in the art. By decrease inpathogenicity, is meant that the antifungal candidate causes at least a10% decrease in the disease caused by contact of the fungal pathogenwith its host, as compared to the disease caused in the absence of theantifungal candidate. Preferably, the disease will be decreased by atleast 40%. More preferably, the disease will be decreased by at least50%, 75% or at least 90% or more. Methods for measuring fungal diseaseare well known to those skilled in the art, and include such metrics aslesion formation, lesion size, sporulation, respiratory failure, and/ordeath.

[0085] The ability of a compound to inhibit homocitrate synthaseactivity can be detected using in vitro enzymatic assays in which thedisappearance of a substrate or the appearance of a product is directlyor indirectly detected. Homocitrate synthase catalyzes the irreversibleor reversible reaction acetyl-CoA and H₂O and2-oxoglutarate=2-hydroxybutane-1,2,4-tricarboxylate and CoA (see FIG.1). Methods for detection of 2-hydroxybutane-1,2,4-tricarboxylate, CoA,acetyl-CoA, and/or 2-oxoglutarate, include spectrophotometry, massspectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

[0086] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising either:

[0087] a) contacting acetyl-CoA and H₂O and 2-oxoglutarate with ahomocitrate synthase;

[0088] b) contacting acetyl-CoA and H₂O and 2-oxoglutarate withhomocitrate synthase and said test compound; and

[0089] c) determining the change in concentration for at least one ofthe following: 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,acetyl-CoA, CoA, and/or H₂O.

[0090] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic. or,

[0091] a) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA with ahomocitrate synthase;

[0092] b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA with ahomocitrate synthase and said test compound; and

[0093] c) determining the change in concentration for at least one ofthe following: 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,acetyl-CoA, CoA, and/or H₂O.

[0094] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0095] Enzymatically active fragments of a fungal homocitrate synthaseare also useful in the methods of the invention. For example, apolypeptide comprising at least 100 consecutive amino acid residues of afungal homocitrate synthase may be used in the methods of the invention.In addition, a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%or at least 98% sequence identity with a fungal homocitrate synthase maybe used in the methods of the invention. Most preferably, thepolypeptide has at least 50% sequence identity with a fungal homocitratesynthase and at least 10%, 25%, 75% or at least 90% of the activitythereof.

[0096] Thus, the invention provides a method for identifying a testcompound as a candidate for a fungicide, comprising:

[0097] a) contacting acetyl-CoA and H₂O and 2-oxoglutarate with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with a homocitrate synthase, apolypeptide having at least 50% sequence identity with a homocitratesynthase and having at least 10% of the activity thereof, and apolypeptide comprising at least 100 consecutive amino acids of ahomocitrate synthase

[0098] b) contacting acetyl-CoA and H₂O and 2-oxoglutarate with saidpolypeptide and said test compound; and

[0099] c) determining the change in concentration for at least one ofthe following: 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,acetyl-CoA, CoA, and/or H₂O.

[0100] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic. or,

[0101] a) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with a homocitrate synthase, apolypeptide having at least 50% sequence identity with a homocitratesynthase and at least 10% of the activity thereof, and a polypeptidecomprising at least 100 consecutive amino acids of a homocitratesynthase

[0102] b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA, withsaid polypeptide and said test compound; and

[0103] c) determining the change in concentration for at least one ofthe following, 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,acetyl-CoA, CoA, and/or H₂O.

[0104] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0105] For the in vitro enzymatic assays, homocitrate synthase proteinand derivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of homocitrate synthase may bedescribed in Jaklitsch and Kubicek (Jaklitsch, W. M. and C. P. Kubicek(1990) Biochem J 269: 247-53 (PMID: 2115771)). Other methods for thepurification of homocitrate synthase proteins and polypeptides are knownto those skilled in the art.

[0106] As an alternative to in vitro assays, the invention also providescell based assays. In one embodiment, the invention provides a methodfor identifying a test compound as a candidate for a antibiotic,comprising:

[0107] a) measuring the expression of a homocitrate synthase in a cell,cells, tissue, or an organism in the absence of said compound;

[0108] b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said homocitrate synthasein said cell, cells, tissue, or organism;

[0109] c) comparing the expression of homocitrate synthase in steps (a)and (b);

[0110] wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.

[0111] Expression of homocitrate synthase can be measured by detectingthe HCS1 primary transcript or mRNA, homocitrate synthase polypeptide,or homocitrate synthase enzymatic activity. Methods for detecting theexpression of RNA and proteins are known to those skilled in the art.See, for example, Current Protocols in Molecular Biology Ausubel et al.,eds., Greene Publishing and Wiley-Interscience, New York, 1995. Themethod of detection is not critical to the invention. Methods fordetecting HCS1 RNA include, but are not limited to amplification assayssuch as quantitative reverse transcriptase-PCR, and/or hybridizationassays such as Northern analysis, dot blots, slot blots, in-situhybridization, transcriptional fusions using a HCS1 promoter fused to areporter gene, DNA assays, and microarray assays.

[0112] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect HCS1protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with HCS1,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

[0113] Chemicals, compounds or compositions identified by the abovemethods as modulators, preferably inhibitors, of HCS1 expression oractivity can then be used to control fungal growth. Diseases such asrusts, mildews, and blights spread rapidly once established. Fungicidesare thus routinely applied to growing and stored crops as a preventivemeasure, generally as foliar sprays or seed dressings. For example,compounds that inhibit fungal growth can be applied to a fungus orexpressed in a fungus, in order to prevent fungal growth. Thus, theinvention provides a method for inhibiting fungal growth, comprisingcontacting a fungus with a compound identified by the methods of theinvention as having antifungal activity.

[0114] Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

[0115] Examples of undesired fungi include, but are not limited toPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

[0116] Also provided is a method of screening for an antibiotic bydetermining whether a test compound is active against the geneidentified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO:3), or the biochemical pathway or pathways it functions on.

[0117] In one particular embodiment, the method is performed byproviding an organism having a first form of the gene corresponding toeither SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutantform, a homologue, or a heterologous HCS1 gene that performs a similarfunction as HCS1. The first form of HCS1 may or may not confer a growthconditional phenotype, i.e., a lysine requiring phenotype, and/or ahypersensitivity or hyposensitivity phenotype on the organism havingthat altered form. In one particular embodiment a mutant form contains atransposon insertion. A comparison organism having a second form of anHCS1, different from the first form of the gene is also provided, andthe two organisms are separately contacted with a test compound. Thegrowth of the two organisms in the presence of the test compound is thencompared.

[0118] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0119] a) providing cells having one form of a homocitrate synthasegene, and providing comparison cells having a different form of ahomocitrate synthase gene,

[0120] b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and comparison cellsin the presence of the test compound,

[0121] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0122] It is recognized in the art that the optional determination ofthe growth of said first organism and said comparison second organism inthe absence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of an HCS1gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

[0123] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry. New York, Worth Publishers).

[0124] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which HCS1 functions, comprising:

[0125] a) providing cells having one form of a gene in the lysinebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene.

[0126] b) contacting said cells and comparison cells with a said testcompound,

[0127] c) determining the growth of said cells and comparison cells inthe presence of said test compound.

[0128] wherein a difference in growth between said cells and saidcomparison cells in the presence of said compound indicates that saidcompound is a candidate for an antibiotic.

[0129] The use of multi-well plates for screening is a format thatreadily accommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

[0130] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger, A., D. Nelson, etal. (1993) Principles of Biochemistry. New York, Worth Publishers).Thus, in one embodiment, the invention provides a method for screeningfor test compounds acting against the biochemical and/or genetic pathwayor pathways in which HCS1 functions, comprising:

[0131] (a) providing paired growth media; comprising a first medium anda second medium, wherein said second medium contains a higher level oflysine than said first medium;

[0132] (b) contacting an organism with said test compound;

[0133] (c) inoculating said first and second media with said organism;and

[0134] (d) determining the growth of said organism;

[0135] wherein a difference in growth of the organism between said firstand second media indicates that said test compound is a candidate for anantibiotic.

[0136] It is recognized in the art that the optional determination ofthe growth of said organism in the paired media in the absence of anytest compounds may be performed to control for any inherent differencesin growth as a result of the different media. Growth and/orproliferation of an organism is measured by methods well known in theart such as optical density measurements, and the like. In a preferredembodiment, the organism is Magnaporthe grisea.

EXPERIMENTAL EXAMPLE 1 Construction of Plasmids with a TransposonContaining a Selectable Marker

[0137] Construction of Sif transposon: Sif was constructed using theGPS3 vector from the GPS-M mutagenesis system from New England Biolabs,Inc. (Beverly, Mass.) as a backbone. This system is based on thebacterial transposon Tn7. The following manipulations were done to GPS3according to Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. The kanamycin resistancegene (npt) contained between the Tn7 arms was removed by EcoRVdigestion. The bacterial hygromycin B phosphotransferase (hph) gene(Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) under controlof the Aspergillus nidulans trpC promoter and terminator (Mullaney etal. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158796)) was cloned by aHpaI/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yieldingpSif1. Excision of the ampicillin resistance gene (bla) from pSif1 wasachieved by cutting pSif1 with XmnI and Bg1I followed by a T4 DNApolymerase treatment to remove the 3′ overhangs left by the Bg1Idigestion and religation of the plasmid to yield pSif. Top 10F′electrocompetent E. coli cells (Invitrogen) were transformed withligation mixture according to manufacturer's recommendations.Transformants containing the Sif transposon were selected on LB agar(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press.) containing 50 ug/ml of hygromycin B(Sigma Chem. Co., St. Louis, Mo.).

EXAMPLE 2 Construction of a Cosmid Library Containing Fungal Genes and aSelectable Marker

[0138] Cosmid libraries were constructed in the pcosKA5 vector (Hamer etal. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) asdescribed in Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. Cosmid libraries werequality checked by pulsed-field gel electrophoresis, restrictiondigestion analysis, and PCR identification of single genes.

EXAMPLE 3 Construction of Cosmids with Transposon Inserted into FungalGenes

[0139] Sif Transposition into a Cosmid: Transposition of Sif into thecosmid framework was carried out as described by the GPS-M mutagenesissystem (New England Biolabs, Inc.). Briefly, 2 ul of the 10×GPS buffer,70 ng of supercoiled pSIF, 8-12 μg of target cosmid DNA were mixed andtaken to a final volume of 20 ul with water. 1 ul of transposase(TnsABC) was added to the reaction and incubated for 10 minutes at 37°C. to allow the assembly reaction to happen. After the assembly reaction1 ul of start solution was added to the tube, mixed well and incubatedfor 1 hour at 37° C. followed by heat inactivation of the proteins at75° C. for 10 min. Destruction of the remaining untransposed pSif wasdone by PISceI digestion at 37° C. for 2 hours followed by 10 minincubation at 75° C. to inactivate the proteins. Transformation of Top10F′ electrocompetent cells (Invitrogen) was done according tomanufacturers recommendations. Sif-containing cosmid transformants wereselected by growth on LB agar plates containing 50 ug/ml of hygromycin B(Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

EXAMPLE 4 High Throughput Preparation and Verification of Insertion ofTransposon into Fungal Genes

[0140]E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37 C overnight. E.coli cells were pelleted by centrifugation and cosmids were isolated bya modified alkaline lysis method (Marra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

[0141] DNA sequences adjacent to the site of the insertion werecollected and used to search DNA and protein databases using the BLASTalgorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402(PMID: 9254694)). A single insertion of SIF into the Magnaporthe griseaHCS1 gene was chosen for further analysis. This construct was designatedcpgmra0023008h04 and it contains the SIF transposon between amino acids334 and 335 relative to the Penicillium chrysogenum homologue (totallength—474 amino acids, GENBANK: PCAJ3630 accession number AJ223630).

EXAMPLE 5 Preparation of Cosmid DNA and Transformation of the FungusMagnaporthe grisea

[0142] Cosmid DNA from the HCS1 transposon tagged cosmid clone wasprepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI(New England Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50 ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set withthe following parameters: resistance 200 ohm, capacitance 25 uF, voltage0.6 kV. Transformed protoplasts were regenerated in complete agar media(CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) withthe addition of 20% sucrose for one day, then overlayed with CM agarmedia containing hygromycin B (250 ug/ml) to select transformants.Transformants were screened for homologous recombination events in thetarget gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98:5110-15 (PMID: 11296265)). Two independent strains were identified andare hereby referred to as KO1-1 and KO1-2, respectively.

EXAMPLE 6 Effect of Transposon Insertion on Magnaporthe Pathogenicity

[0143] The target fungal strains, KO1-1 and KO1-2, obtained in Example 5and the wild type strain, Guy11, were subjected to a pathogenicity assayto observe infection over a 1-week period. Rice infection assays wereperformed using Indian rice cultivar CO39 essentially as described inValent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All threestrains were grown for spore production on complete agar media. Sporeswere harvested and the concentration of spores adjusted for whole plantinoculations. Two-week-old seedlings of cultivar C039 were sprayed with12 ml of conidial suspension (5×10⁴ conidia per ml in 0.01% Tween-20(Polyoxyethylensorbitan monolaureate) solution). The inoculated plantswere incubated in a dew chamber at 27° C. in the dark for 36 hours, andtransferred to a growth chamber (27° C. 12 hours/21° C. 12 hours 70%humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5,and 7 days post-inoculation and examined for signs of successfulinfection (i.e. lesions). FIG. 2 shows the effects of HCS1 genedisruption on Magnaporthe infection at five days post-inoculation.

EXAMPLE 7 Verification of Gene Function by Analysis of NutritionalRequirements

[0144] The fungal strains, KO1-1 and KO1-2, containing the HCS1disrupted gene obtained in Example 5 were analyzed for their nutritionalrequirement for lysine using the PM5 phenotype microarray from Biolog,Inc. (Hayward, Calif.). The PM5 plate tests for the auxotrophicrequirement for 94 different metabolites. The inoculating fluid consistsof 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO₃, 6.7 mMKCl, 3.5 mM Na₂SO₄, 11 mM KH₂PO₄, 0.01% p-iodonitrotetrazolium violet,0.1 mM MgCl₂, 1.0 mM CaCl₂ and trace elements. Final concentrations oftrace elements are: 7.6 μM ZnCl₂, 2.5 μM MnCl₂.4H₂O, 1.8 μM FeCl₂.4H₂O,0.71 μM COCl₂.6H2O, 0.64 μM CuCl₂.2H₂O, 0.62 μM Na₂MoO₄, 18 μM H₃BO₃. pHadjusted to 6.0 with NaOH. Spores for each strain were harvested intothe inoculating fluid. The spore concentrations were adjusted to 2×10⁵spores/ml. 100 μl of spore suspension were deposited into each well ofthe microtiter plates. The plates were incubated at 25° C. for 7 days.Optical density (OD) measurements at 490 nm and 750 nm were taken daily.The OD₄₉₀ measures the extent of tetrazolium dye reduction and the levelof growth, and OD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Dataconfirming the annotated gene function is presented as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of L-lysine.

EXAMPLE 8 Cloning and Expression Strategies, Extraction and Purificationof the Homocitrate Synthase Protein

[0145] The following protocol may be employed to obtain the purified thehomocitrate synthase protein.

[0146] Cloning and Expression Strategies:

[0147] A HCS1 cDNA gene can be cloned into E. coli (pETvectors-Novagen),

[0148] Baculovirus (Pharmingen) and Yeast (Invitrogen) expressionvectors containing His/fusion protein tags. Evaluate the expression ofrecombinant protein by SDS-PAGE and Western blot analysis.

[0149] Extraction:

[0150] Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer By sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

[0151] Purification:

[0152] Purify recombinant protein by Ni-NTA affinity chromatography(Qiagen).

[0153] Purification protocol: perform all steps at 4° C.:

[0154] Use 3 ml Ni-beads (Qiagen)

[0155] Equilibrate column with the buffer

[0156] Load protein extract

[0157] Wash with the equilibration buffer

[0158] Elute bound protein with 0.5 M imidazole

EXAMPLE 9 Assays for Testing Binding of Test Compounds to HomocitrateSynthase

[0159] The following protocol may be employed to identify test compoundsthat bind to the homocitrate synthase protein.

[0160] Purified full length homocitrate synthase polypeptide with aHis/fusion protein tag (Example 8) is bound to a HisGrab Nickel CoatedPlate (Pierce, Rockford, Ill.) following manufacturer's instructions.

[0161] Buffer conditions are optimized (e.g. ionic strength or pH,Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53 (PMID:2115771)) for binding of radiolabeled (acetyl-1-¹⁴C)-coenzyme A(Sigma-Aldrich Co.) to the bound HCS1.

[0162] Screening of test compounds is performed by adding test compoundand (acetyl-1-¹⁴C)-coenzyme A (Sigma-Aldrich Co.) to the wells of theHisGrab™ plate containing bound HCS1.

[0163] The wells are washed to remove excess labeled ligand andscintillation fluid (Scintiverse®, Fisher Scientific) is added to eachwell.

[0164] The plates are read in a microplate scintillation counter.

[0165] Candidate compounds are identified as wells with lowerradioactivity as compared to control wells with no test compound added.

[0166] Additionally, a purified polypeptide comprising 10-50 amino acidsfrom the M. grisea homocitrate synthase is screened in the same way. Apolypeptide comprising 10-50 amino acids is generated by subcloning aportion of the HCS1 gene into a protein expression vector that adds aHis-Tag when expressed (see Example 8). Oligonucleotide primers aredesigned to amplify a portion of the HCS1 gene using the polymerasechain reaction amplification method. The DNA fragment encoding apolypeptide of 10-50 amino acids is cloned into an expression vector,expressed in a host organism and purified as described in Example 8above.

[0167] Test compounds that bind HCS1 are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al. (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

EXAMPLE 10 Assays for Testing Inhibitors or Candidates for Inhibition ofHomocitrate Synthase Activity

[0168] The enzymatic activity of homocitrate synthase is determined inthe presence and absence of candidate compounds in a suitable reactionmixture, such as described by Gray and Bhattacharjee (Gray, G S andBhattacharjee, J K (1976) Can J Microbiol 22: 1664-7 (PMID: 10066)), orJaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53 (PMID:2115771). Candidate compounds are identified when a decrease in productsor a lack of decrease in substrates is detected with the reactionproceeding in either direction.

[0169] Additionally, the enzymatic activity of a polypeptide comprising10-50 amino acids from the M. grisea homocitrate synthase is determinedin the presence and absence of candidate compounds in a suitablereaction mixture, such as described by Gray and Bhattacharjee (Gray, G Sand Bhattacharjee, J K (1976) Can J Microbiol 22: 1664-7 (PMID: 10066)),or Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53(PMID: 2115771). A polypeptide comprising 10-50 amino acids is generatedby subcloning a portion of the HCS1 gene into a protein expressionvector that adds a His-Tag when expressed (see Example 8).Oligonucleotide primers are designed to amplify a portion of the HCS1gene using polymerase chain reaction amplification method. The DNAfragment encoding a polypeptide of 10-50 amino acids is cloned into anexpression vector, expressed and purified as described in Example 8above.

[0170] Test compounds identified as inhibitors of HCS1 activity arefurther tested for antibiotic activity. Magnaporthe grisea fungal cellsare grown under standard fungal growth conditions that are well knownand described in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et al. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

EXAMPLE 11 Assays for Testing Compounds or Candidates for Compounds ThatAlter the Expression of the Homocitrate Synthase Gene

[0171]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on completeagar or oatmeal agar media after growth for 10-13 days in the light at25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml. 25 mlcultures are prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the HCS1 gene as a probe. Testcompounds resulting in a reduced level of HCS1 mRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

EXAMPLE 12 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Homocitrate Synthase with No Activity

[0172]Magnaporthe grisea fungal cells containing a mutant form of theHCS1 gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-lysine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium containing 100 μML-lysine to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores are added to each well of 96-well plates to which a test compoundis added (at varying concentrations). The total volume in each well is200 μl. Wells with no test compound present (growth control), and wellswithout cells are included as controls (negative control). The platesare incubated at 25° C. for seven days and optical density measurementsat 590 nm are taken daily. Wild type cells are screened under the sameconditions. The effect of each compound on the mutant and wild-typefungal strains is measured against the growth control and the percent ofinhibition is calculated as the OD₅₉₀ (fungal strain plus testcompound)/OD₅₉₀ (growth control)×100. The percent of growth inhibitionas a result of a test compound on a fungal strain and that on the wildtype cells are compared. Compounds that show differential growthinhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26:177-221 (PMID: 7749303)).

EXAMPLE 13 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Homocitrate Synthase with ReducedActivity

[0173]Magnaporthe grisea fungal cells containing a mutant form of theHCS1 gene, such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 4mM L-lysine (Sigma-Aldrich Co.) after growth for 10-13 days in the lightat 25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml.Approximately 4×10⁴ spores are added to each well of 96-well plates towhich a test compound is added (at varying concentrations). The totalvolume in each well is 200%1. Wells with no test compound present(growth control), and wells without cells are included as controls(negative control). The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. Wild typecells are screened under the same conditions. The effect of eachcompound on the mutant and wild-type fungal strains is measured againstthe growth control and the percent of inhibition is calculated as theOD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growth control)×100. Thepercent of growth inhibition as a result of a test compound on a fungalstrain and that on the wild-type cells are compared. Compounds that showdifferential growth inhibition between the mutant and the wild type areidentified as potential antifungal compounds. Similar protocols may befound in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:7749303)).

EXAMPLE 14 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a Lysine Biosynthetic Gene with NoActivity

[0174]Magnaporthe grisea fungal cells containing a mutant form of a genein the lysine biosynthetic pathway (e.g. L-Aminoadipate-semialdehydedehydrogenase (E.C. 1.2.1.31)) are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 4 mM L-lysine (Sigma-Aldrich Co.) after growth for 10-13 daysin the light at 25° C. using a moistened cotton swab. The concentrationof spores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium containing 100 μM L-lysine to aconcentration of 2×10⁵ spores per ml. Approximately 4×10⁴ spores orcells are harvested and added to each well of 96-well plates to whichgrowth media is added in addition to an amount of test compound (atvarying concentrations). The total volume in each well is 200 μl. Wellswith no test compound present, and wells without cells are included ascontrols. The plates are incubated at 25° C. for seven days and opticaldensity measurements at 590 nm are taken daily. Wild type cells arescreened under the same conditions. The effect of each compound on themutant and wild-type fungal strains is measured against the growthcontrol and the percent of inhibition is calculated as the OD₅₉₀ (fungalstrain plus test compound)/OD₅₉₀ (growth control)×100. The percent ofgrowth inhibition as a result of a test compound on a fungal strain andthat on the wild type cells are compared. Compounds that showdifferential growth inhibition between the mutant and the wild-type areidentified as potential antifungal compounds. Similar protocols may befound in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:7749303)).

EXAMPLE 15 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a Lysine Biosynthetic Gene withReduced Activity

[0175]Magnaporthe grisea fungal cells containing a mutant form of a genein the lysine biosynthetic pathway (e.g. L-Aminoadipate-semialdehydedehydrogenase (E.C. 1.2.1.31)), such as a promoter truncation thatreduces expression, are grown under standard fungal growth conditionsthat are well known and described in the art. A promoter truncation ismade by deleting a portion of the promoter upstream of the transcriptionstart site using standard molecular biology techniques that are wellknown and described in the art (Sambrook et al (1989) Molecular Cloning,a Laboratory Manual, Cold Spring Harbor Laboratory Press). Magnaporthegrisea fungal cells containing a mutant form of are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-lysine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26:177-221 (PMID: 7749303)).

EXAMPLE 16 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Fungal HCS1 and a Second Fungal Strain Containing aHeterologous HCS1 Gene

[0176] Wild-type Magnaporthe grisea fungal cells and M. grisea fungalcells lacking a functional HCS1 gene and containing a HCS1 gene fromThermus aquaticus (Genbank accession 087198, 56% sequence identity) aregrown under standard fungal growth conditions that are well known anddescribed in the art. A M. grisea strain carrying a heterologous HCS1gene is made as follows:

[0177] A M. grisea strain is made with a nonfunctional HCS1 gene, suchas one containing a transposon insertion in the native gene (seeExamples 4 and 5).

[0178] A construct containing a heterologous HCS1 gene is made bycloning the HCS1 gene from Thermus aquaticus into a fungal expressionvector containing a trpC promoter and terminator (e.g. pCB1003, Carrollet al. (1994) Fungal Gen News Lett 41: 22) using standard molecularbiology techniques that are well known and described in the art(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press).

[0179] The said construct is used to transform the M. grisea strainlacking a functional HCS1 gene (see Example 5). Transformants areselected on minimal agar medium lacking L-lysine. Only transformantscarrying a functional HCS1 gene will grow.

[0180] Wild-type strains of Magnaporthe grisea and strains containing aheterologous form of HCS1 are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumafter growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores or cells are harvested and added to each well of 96-well platesto which growth media is added in addition to an amount of test compound(at varying concentrations). The total volume in each well is 200 μl.Wells with no test compound present, and wells without cells areincluded as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The effectof each compound on the wild-type and heterologous fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on the wild-type and heterologous fungal strains are compared.Compounds that show differential growth inhibition between the wild-typeand heterologous strains are identified as potential antifungalcompounds with specificity to the native or heterologous HCS1 geneproducts. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 17 Pathway Specific In Vivo Assay Screening Protocol

[0181]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on oatmealagar media after growth for 10-13 days in the light at 25° C. using amoistened cotton swab. The concentration of spores is determined using ahemocytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 4 mM L-lysine(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 4 mM L-lysine. Test compounds are added to wellscontaining spores in minimal media and minimal media containingL-lysine. The total volume in each well is 200 μl. Both minimal mediaand L-lysine containing media wells with no test compound are providedas controls. The plates are incubated at 25° C. for seven days andoptical density measurements at 590 nm are taken daily. A compound isidentified as a candidate for an antibiotic acting against the lysinebiosynthetic pathway when the observed growth in the well containingminimal media is less than the observed growth in the well containingL-lysine as a result of the addition of the test compound. Similarprotocols may be found in Kirsch and DiDomenico ((1994) Biotechnology26: 177-221 (PMID: 7749303)).

[0182] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may be made and still fall within the scopeof the invention. The foregoing examples are intended to exemplifyvarious specific embodiments of the invention and do not limit its scopein any manner.

1 3 1 1458 DNA Magnaporthe grisea 1 atgtgcccat cctgcgagcc tgagcaagccgctgcctcca atggcaatgc gaacggcaat 60 ggcgcctcca atggcaatgg aaaccacgacggaatgactg gtattgagac tcgccaagca 120 caaaacgcac gctaccagcc atcacggaatccctaccagc ccgtcggtga ctttttgtcc 180 aacgtgaaca acttcaagat cattgagagcaccctgcgag agggcgagca gttcgccaat 240 gccttcttcg acacggccaa gaagattgagatcgccaagg cgctggacga ctttggcgtc 300 gactacatcg agctcaccag cccggctgcctcggagcagt ccaggcttga ctgcgctgcc 360 atctgcaagc tgggactcaa ggccaagatcctcacccaca tcaggtgcca catggacgac 420 gcgcgcatcg ccgtcgagac cggtgttgacggcgtcgaca ttgtcatcgg cacctcttcg 480 ttcctcatgg agcactcgca cggcaaggacatgacctaca tcaccaacac ggccattgag 540 gtcatcaact ttgtcaagag caagggcatcgaggtccgct tctcatccga ggactcgttc 600 cgcagcaacc tggttgacct gctgagcatctactcgaccg tcgacaagat tggtgtcaac 660 cgtgtcggta ttgctgatac cgtcggttgcgcctcgcccc gccaggtcta cgacctggtc 720 aagaccctgc gtggtgttgt ctcttgtgacattgagacac acttccacaa cgacactggc 780 tgtgccatct caaatgcttt ttgcgctttggaggccggtg ctacccacat cgacacgtgt 840 gtcctgggta tcggcgagcg taacggaattacccctcttg gaggtctgat ggctcgcatg 900 attgtcggct ccaaggacta cgttctgagcaagtacaagc tgcacaagct caaggacatt 960 gaggagcttg ttgccgacgc cgttcaggtcaacattcctt tcaataacta catcactggt 1020 ttctgtgctt tcacccacaa ggccggtatccatgccaagg ctattctcaa gaacccctca 1080 acatatgaga ttattgaccc gactctgttcggcatcactc gctatgtcca cttcgccagc 1140 agattgacgg gatggaacgc aatcaagagcagagcatcgc agctcaacat tgagatgacg 1200 gatgagcagt gcaaggagtg cactgccaagatcaagctgt tggctgacat taggccgatc 1260 gctatcgacg acgccgactc catcattcacgcattccacc gcagcatcaa ctcgggccag 1320 cctattcagt ctctcggaag cctgctccccaacatgacgg aggaggagaa ggccgccctg 1380 gcagatgtag agcgccgtga gtcgaacgatgccgagcaac cggcggccaa gagggccaag 1440 gtcgaggctg ttgcatga 1458 2 2346DNA Magnaporthe grisea exon (130)..(384) 2 caaagctgga gctccaccgcggtggcggcc gctctagaac tagtggatcc cccgggctgc 60 aggaattcgg cacgagccaagtcccagcct cccattgagc tttactcaca acaatcccaa 120 accaccaaa atg tgc ccatcc tgc gag cct gag caa gcc gct gcc tcc aat 171 Met Cys Pro Ser Cys GluPro Glu Gln Ala Ala Ala Ser Asn 1 5 10 ggc aat gcg aac ggc aat ggc gcctcc aat ggc aat gga aac cac gac 219 Gly Asn Ala Asn Gly Asn Gly Ala SerAsn Gly Asn Gly Asn His Asp 15 20 25 30 gga atg act ggt att gag act cgccaa gca caa aac gca cgc tac cag 267 Gly Met Thr Gly Ile Glu Thr Arg GlnAla Gln Asn Ala Arg Tyr Gln 35 40 45 cca tca cgg aat ccc tac cag ccc gtcggt gac ttt ttg tcc aac gtg 315 Pro Ser Arg Asn Pro Tyr Gln Pro Val GlyAsp Phe Leu Ser Asn Val 50 55 60 aac aac ttc aag atc att gag agc acc ctgcga gag ggc gag cag ttc 363 Asn Asn Phe Lys Ile Ile Glu Ser Thr Leu ArgGlu Gly Glu Gln Phe 65 70 75 gcc aat gcc ttc ttc gac acg gtgagtcaagccacatcgca agcaaatact 414 Ala Asn Ala Phe Phe Asp Thr 80 85 tgctcctcacaacggccgca agcctgggct actttggtag ctcggcggtg tttttgctgt 474 cgatgtgtcctggcggcatc ccggcgcaaa aacagacctc atagactgac tcatgctttt 534 tttaacctccgcgcag gcc aag aag att gag atc gcc aag gcg ctg gac gac 586 Ala Lys LysIle Glu Ile Ala Lys Ala Leu Asp Asp 90 95 ttt ggc gtc gac tac atc gagctc acc agc ccg gct gcc tcg gag cag 634 Phe Gly Val Asp Tyr Ile Glu LeuThr Ser Pro Ala Ala Ser Glu Gln 100 105 110 tcc agg ctt gac tgc gct gccatc tgc aag ctg gga ctc aag gcc aag 682 Ser Arg Leu Asp Cys Ala Ala IleCys Lys Leu Gly Leu Lys Ala Lys 115 120 125 atc ctc acc cac atc agg tgccac atg gac gac gcg cgc atc gcc gtc 730 Ile Leu Thr His Ile Arg Cys HisMet Asp Asp Ala Arg Ile Ala Val 130 135 140 145 gag acc ggt gtt gac ggcgtc gac att gtc atc ggc acc tct tcg ttc 778 Glu Thr Gly Val Asp Gly ValAsp Ile Val Ile Gly Thr Ser Ser Phe 150 155 160 ctc atg gag cac tcg cacggc aag gac atg acc tac atc acc aac acg 826 Leu Met Glu His Ser His GlyLys Asp Met Thr Tyr Ile Thr Asn Thr 165 170 175 gcc att gag gtc atc aacttt gtc aag agc aag ggc atc gag gtc cgc 874 Ala Ile Glu Val Ile Asn PheVal Lys Ser Lys Gly Ile Glu Val Arg 180 185 190 ttc tca tcc gag gac tcgttc cgc agc aac ctg gtt gac ctg ctg agc 922 Phe Ser Ser Glu Asp Ser PheArg Ser Asn Leu Val Asp Leu Leu Ser 195 200 205 atc tac tcg acc gtc gacaag att ggt gtc aac cgt gtc ggt att gct 970 Ile Tyr Ser Thr Val Asp LysIle Gly Val Asn Arg Val Gly Ile Ala 210 215 220 225 gat acc gtc ggt tgcgcc tcg ccc cgc cag gtc tac gac ctg gtc aag 1018 Asp Thr Val Gly Cys AlaSer Pro Arg Gln Val Tyr Asp Leu Val Lys 230 235 240 acc ctg cgt ggt gttgtc tct tg gtgagccaca ggtctgatga atcttgtgct 1071 Thr Leu Arg Gly Val ValSer Cys 245 gcttggtgct gatgctaaca gttcgatag t gac att gag aca cac ttccac aac 1125 Asp Ile Glu Thr His Phe His Asn 250 255 gac act ggc tgt gccatc tca aat gct ttt tgc gct ttg gag gcc ggt 1173 Asp Thr Gly Cys Ala IleSer Asn Ala Phe Cys Ala Leu Glu Ala Gly 260 265 270 gct acc cac atc gacacg tgt gtc ctg ggt atc ggc gag cgt aac gga 1221 Ala Thr His Ile Asp ThrCys Val Leu Gly Ile Gly Glu Arg Asn Gly 275 280 285 att acc cct ctt ggaggt ctg atg gct cgc atg att gtc ggc tcc aag 1269 Ile Thr Pro Leu Gly GlyLeu Met Ala Arg Met Ile Val Gly Ser Lys 290 295 300 305 gac tac gtt ctgagc aag tac aag ctg cac aag ctc aag gac att gag 1317 Asp Tyr Val Leu SerLys Tyr Lys Leu His Lys Leu Lys Asp Ile Glu 310 315 320 gag ctt gtt gccgac gcc gtt cag gtc aac at gtaagttttg ccatcccagt 1369 Glu Leu Val AlaAsp Ala Val Gln Val Asn Ile 325 330 gcagttttca ttctgggtag gattgctaacattttgtctc tgtag t cct ttc aat 1424 Pro Phe Asn 335 aac tac atc act ggtttc tgt gct ttc acc cac aa gtatgttccg 1469 Asn Tyr Ile Thr Gly Phe CysAla Phe Thr His Lys 340 345 tcacacactg gtatctacta ttgattcaaa actaactcgttgctatag g gcc ggt 1524 Ala Gly atc cat gcc aag gct att ctc aag aac ccctca aca tat gag att att 1572 Ile His Ala Lys Ala Ile Leu Lys Asn Pro SerThr Tyr Glu Ile Ile 350 355 360 365 gtatgttttt gatctgttca cgcactgtgccagcatgggt atgatgagcc gaaaatacta 1632 acccttgatt aatcag gac ccg act ctgttc ggc atc act cgc tat gtc cac 1684 Asp Pro Thr Leu Phe Gly Ile Thr ArgTyr Val His 370 375 ttc gcc agc aga ttg acg gga tgg aac gca atc aag agcaga gca tcg 1732 Phe Ala Ser Arg Leu Thr Gly Trp Asn Ala Ile Lys Ser ArgAla Ser 380 385 390 cag ctc aac att gag atg acg gat gag cag tgc aag gagtgc act gcc 1780 Gln Leu Asn Ile Glu Met Thr Asp Glu Gln Cys Lys Glu CysThr Ala 395 400 405 aag atc aag ctg ttg gct gac att agg ccg atc gct atcgac gac gcc 1828 Lys Ile Lys Leu Leu Ala Asp Ile Arg Pro Ile Ala Ile AspAsp Ala 410 415 420 425 gac tcc atc att cac gca ttc cac cgc agc atc aactcg ggc cag cct 1876 Asp Ser Ile Ile His Ala Phe His Arg Ser Ile Asn SerGly Gln Pro 430 435 440 att cag tat ctc gga agc ctg ctc ccc aac atg acggag gag gag aag 1924 Ile Gln Tyr Leu Gly Ser Leu Leu Pro Asn Met Thr GluGlu Glu Lys 445 450 455 gcc gcc ctg gca gat gta gag cgc cgt gag tcg aacgat gcc gag caa 1972 Ala Ala Leu Ala Asp Val Glu Arg Arg Glu Ser Asn AspAla Glu Gln 460 465 470 ccg gcg gcc aag agg gcc aag gtc gag gct gtt gcat gagcacaacg 2019 Pro Ala Ala Lys Arg Ala Lys Val Glu Ala Val Ala 475480 485 gaatttttga gcattgtcga agcgtgagcg agtcacatat atattgttgacgcagaattt 2079 tggtggtcaa agggaagtac agaaaggcct tgggctttga ttttcctaaccccaaagcgt 2139 tgacattttt attatgtctt cttctgtctg catacgaagt caaaaaaggaaggagaaagg 2199 aaaagtatcg tcaggatggg atggtttacg gatctacatt ggtaccggagctattcaagg 2259 atagattgtg tttgctttga tttgccccca tggatgagtt ggggccttttgctgatttgc 2319 tatatgttgc tataccattg aatgaaa 2346 3 449 PRT Magnaporthegrisea 3 Met Cys Pro Ser Cys Glu Pro Glu Gln Ala Ala Ala Ser Asn Gly Asn1 5 10 15 Ala Asn Gly Asn Gly Ala Ser Asn Gly Asn Gly Asn His Asp GlyMet 20 25 30 Thr Gly Ile Glu Thr Arg Gln Ala Gln Asn Ala Arg Tyr Gln ProSer 35 40 45 Arg Asn Pro Tyr Gln Pro Val Gly Asp Phe Leu Ser Asn Val AsnAsn 50 55 60 Phe Lys Ile Ile Glu Ser Thr Leu Arg Glu Gly Glu Gln Phe AlaAsn 65 70 75 80 Ala Phe Phe Asp Thr Ala Lys Lys Ile Glu Ile Ala Lys AlaLeu Asp 85 90 95 Asp Phe Gly Val Asp Tyr Ile Glu Leu Thr Ser Pro Ala AlaSer Glu 100 105 110 Gln Ser Arg Leu Asp Cys Ala Ala Ile Cys Lys Leu GlyLeu Lys Ala 115 120 125 Lys Ile Leu Thr His Ile Arg Cys His Met Asp AspAla Arg Ile Ala 130 135 140 Val Glu Thr Gly Val Asp Gly Val Asp Ile ValIle Gly Thr Ser Ser 145 150 155 160 Phe Leu Met Glu His Ser His Gly LysAsp Met Thr Tyr Ile Thr Asn 165 170 175 Thr Ala Ile Glu Val Ile Asn PheVal Lys Ser Lys Gly Ile Glu Val 180 185 190 Arg Phe Ser Ser Glu Asp SerPhe Arg Ser Asn Leu Val Asp Leu Leu 195 200 205 Ser Ile Tyr Ser Thr ValAsp Lys Ile Gly Val Asn Arg Val Gly Ile 210 215 220 Ala Asp Thr Val GlyCys Ala Ser Pro Arg Gln Val Tyr Asp Leu Val 225 230 235 240 Lys Thr LeuArg Gly Val Val Ser Cys Asp Ile Glu Thr His Phe His 245 250 255 Asn AspThr Gly Cys Ala Ile Ser Asn Ala Phe Cys Ala Leu Glu Ala 260 265 270 GlyAla Thr His Ile Asp Thr Cys Val Leu Gly Ile Gly Glu Arg Asn 275 280 285Gly Ile Thr Pro Leu Gly Gly Leu Met Ala Arg Met Ile Val Gly Ser 290 295300 Lys Asp Tyr Val Leu Ser Lys Tyr Lys Leu His Lys Leu Lys Asp Ile 305310 315 320 Glu Glu Leu Val Ala Asp Ala Val Gln Val Asn Ile Pro Phe AsnAsn 325 330 335 Tyr Ile Thr Gly Phe Cys Ala Phe Thr His Lys Ala Gly IleHis Ala 340 345 350 Lys Ala Ile Leu Lys Asn Pro Ser Thr Tyr Glu Ile IleAsp Pro Thr 355 360 365 Leu Phe Gly Ile Thr Arg Tyr Val His Phe Ala SerArg Leu Thr Gly 370 375 380 Trp Asn Ala Ile Lys Ser Arg Ala Ser Gln LeuAsn Ile Glu Met Thr 385 390 395 400 Asp Glu Gln Cys Lys Glu Cys Thr AlaLys Ile Lys Leu Leu Ala Asp 405 410 415 Ile Arg Pro Ile Ala Ile Asp AspAla Asp Ser Ile Ile His Ala Phe 420 425 430 His Arg Ser Ile Asn Ser GlyGln Pro Ile Gln Ser Leu Gly Ser Leu 435 440 445 Leu

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting a homocitratesynthase polypeptide with said test compound; and b) detecting thepresence or absence of binding between said test compound and saidhomocitrate synthase polypeptide; wherein binding indicates that saidtest compound is a candidate for an antibiotic.
 2. The method of claim1, wherein said homocitrate synthase polypeptide is a fungal homocitratesynthase polypeptide.
 3. The method of claim 1, wherein said homocitratesynthase polypeptide is a Magnaporthe homocitrate synthase polypeptide.4. The method of claim 1, wherein said homocitrate synthase polypeptideis SEQ ID NO:
 3. 5. A method for determining whether a compoundidentified as an antibiotic candidate by the method of claim 1 hasantifungal activity, further comprising: contacting a fungus or fungalcells with said antibiotic candidate and detecting the decrease ingrowth, viability, or pathogenicity of said fungus or fungal cells.
 6. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: a) contacting said test compound with at least onepolypeptide selected from the group consisting of: a polypeptide havingat least ten consecutive amino acids of a fungal homocitrate synthase, apolypeptide having at least 50% sequence identity with a fungalhomocitrate synthase, and a polypeptide having at least 10% of theactivity thereof, and b) detecting the presence and/or absence ofbinding between said test compound and said polypeptide; wherein bindingindicates that said test compound is a candidate for an antibiotic.
 7. Amethod for determining whether a compound identified as an antibioticcandidate by the method of claim 6 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 8. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting acetyl-CoA and H₂O and 2-oxoglutarate with ahomocitrate synthase; b) contacting acetyl-CoA and H₂O and2-oxoglutarate with a homocitrate synthase and said test compound; andc) determining the change in concentration for at least one of thefollowing: 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,acetyl-CoA, CoA, and/or H₂O; wherein a change in concentration for anyof the above substances between steps (a) and (b) indicates that saidtest compound is a candidate for an antibiotic.
 9. The method of claim8, wherein said homocitrate synthase is a fungal homocitrate synthase.10. The method of claim 8, wherein said homocitrate synthase is aMagnaporthe homocitrate synthase.
 11. The method of claim 8, whereinsaid homocitrate synthase is SEQ ID NO:
 3. 12. A method for determiningwhether a compound identified as an antibiotic candidate by the methodof claim 8 has antifungal activity, further comprising: contacting afungus or fungal cells with said antibiotic candidate and detecting adecrease in growth, viability, or pathogenicity of said fungus or fungalcells.
 13. A method for identifying a test compound as a candidate foran antibiotic, comprising: a) contacting2-hydroxybutane-1,2,4-tricarboxylate and CoA with a homocitratesynthase; b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoAwith a homocitrate synthase and said test compound; and c) determiningthe change in concentration for at least one of the following:2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA,and/or H₂O; wherein a change in concentration for any of the abovesubstances between steps (a) and (b) indicates that said test compoundis a candidate for an antibiotic.
 14. The method of claim 13, whereinsaid homocitrate synthase is a fungal homocitrate synthase.
 15. Themethod of claim 13, wherein said homocitrate synthase is a Magnaporthehomocitrate synthase.
 16. The method of claim 13, wherein saidhomocitrate synthase is SEQ ID NO:
 3. 17. A method for determiningwhether a compound identified as an antibiotic candidate by the methodof claim 13 has antifungal activity, further comprising: contacting afungus or fungal cells with said antibiotic candidate and detecting adecrease in growth, viability, or pathogenicity of said fungus or fungalcells.
 18. A method for identifying a test compound as a candidate foran antibiotic, comprising: a) contacting acetyl-CoA and H₂O and2-oxoglutarate with a polypeptide selected from the group consisting of:a polypeptide having at least 50% sequence identity with a homocitratesynthase, a polypeptide having at least 50% sequence identity with ahomocitrate synthase and having at least 10% of the activity thereof,and a polypeptide comprising at least 100 consecutive amino acids of ahomocitrate synthase b) contacting acetyl-CoA and H₂O and 2-oxoglutaratewith said polypeptide and said test compound; and c) determining thechange in concentration for at least one of the following:2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA,and/or H₂O; wherein a change in concentration for any of the abovesubstances between steps (a) and (b) indicates that said test compoundis a candidate for an antibiotic.
 19. A method for identifying a testcompound as a candidate for an antibiotic, comprising: a) contacting2-hydroxybutane-1,2,4-tricarboxylate and CoA with a polypeptide selectedfrom the group consisting of: a polypeptide having at least 50% sequenceidentity with a homocitrate synthase, a polypeptide having at least 50%sequence identity with a homocitrate synthase and at least 10% of theactivity thereof, and a polypeptide comprising at least 100 consecutiveamino acids of a homocitrate synthase b) contacting2-hydroxybutane-1,2,4-tricarboxylate and CoA, with said polypeptide andsaid test compound; and c) determining the change in concentration forat least one of the following: 2-hydroxybutane-1,2,4-tricarboxylate,2-oxoglutarate, acetyl-CoA, CoA, and/or H₂O; wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 20.A method for identifying a test compound as a candidate for anantibiotic, comprising: a) measuring the expression of a homocitratesynthase in a cell, cells, tissue, or an organism in the absence of saidcompound; b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said homocitrate synthasein said fungus or fungal cell; c) comparing the expression ofhomocitrate synthase in steps (a) and (b); wherein a lower expression inthe presence of said test compound indicates that said compound is acandidate for an antibiotic.
 21. The method of claim 20 wherein said acell, cells, tissue, or organism is, or is derived from a fungus. 22.The method of claim 20 wherein said cell, cells, tissue, or organism is,or is derived from a Magnaporthe fungus or fungal cell.
 23. The methodof claim 20, wherein said homocitrate synthase is SEQ ID NO:
 3. 24. Themethod of claim 20, wherein the expression of homocitrate synthase ismeasured by detecting HCS1 mRNA.
 25. The method of claim 20, wherein theexpression of homocitrate synthase is measured by detecting homocitratesynthase polypeptide.
 26. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) providing cells having oneform of a homocitrate synthase gene, and providing comparison cellshaving a different form of a homocitrate synthase gene, b) contactingsaid cells and said comparison cells with a test compound anddetermining the growth of said cells and comparison cells in thepresence of the test compound; wherein a difference in growth betweensaid cells and said comparison cells in the presence of said compoundindicates that said compound is a candidate for an antibiotic.
 27. Themethod of claim 26 wherein the cells are fungal cells.
 28. The method ofclaim 26 wherein the cells are Magnaporthe cells.
 29. The method ofclaim 26 wherein said form and said comparison form of the homocitratesynthase are fungal homocitrate synthases.
 30. The method of claim 26,wherein at least one form is a Magnaporthe homocitrate synthase.
 31. Themethod of claim 26 wherein said form and said comparison form of thehomocitrate synthase are non-fungal homocitrate synthases.
 32. Themethod of claim 26 wherein one form of the homocitrate synthase is afungal homocitrate synthase, and the other form is a non-fungalhomocitrate synthase.
 33. A method for identifying a test compound as acandidate for an antibiotic, comprising: d) providing cells having oneform of a gene in the lysine biochemical and/or genetic pathway andproviding comparison cells having a different form of said gene. e)contacting said cells and comparison cells with a said test compound, f)determining the growth of said cells and comparison cells in thepresence of said test compound; wherein a difference in growth betweensaid cells and said comparison cells in the presence of said compoundindicates that said compound is a candidate for an antibiotic.
 34. Themethod of claim 33 wherein the cells are fungal cells.
 35. The method ofclaim 33 wherein the cells are Magnaporthe cells.
 36. The method ofclaim 33 wherein said form and said comparison form of the lysinebiosynthesis gene are fungal lysine biosynthesis genes.
 37. The methodof claim 33, wherein at least one form is a Magnaporthe lysinebiosynthesis gene.
 38. The method of claim 33 wherein said form and saidcomparison form of the lysine biosynthesis genes are non-fungal lysinebiosynthesis genes.
 39. The method of claim 33 wherein one form of thelysine biosynthesis gene is a fungal lysine biosynthesis gene, and theother form is a non-fungal lysine biosynthesis gene.
 40. A method fordetermining whether a test compound identified as an antibioticcandidate by the method of claim 33 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 41. A method foridentifying a test compound as a candidate for an antibiotic,comprising: (a) providing paired growth media; comprising a first mediumand a second medium, wherein said second medium contains a higher levelof lysine than said first medium; (b) contacting an organism with saidtest compound; (c) inoculating said first and second media with saidorganism; and (d) determining the growth of said organism; wherein adifference in growth of the organism between said first and second mediaindicates that said test compound is a candidate for an antibiotic. 42.The method of claim 41, wherein said organism is a fungus.
 43. Themethod of claim 41, wherein said organism is Magnaporthe.
 44. Anisolated polynucleotide comprising a nucleotide sequence that encodes apolypeptide of SEQ ID NO:
 3. 45. The polynucleotide of claim 44comprising the nucleotide sequence of SEQ ID NO:
 1. 46. An expressioncassette comprising the polynucleotide of claim
 45. 47. The isolatedpolynucleotide of claim 44 comprising a nucleotide sequence of at least50 to at least 95% sequence identity to SEQ ID NO:
 1. 48. A polypeptideconsisting essentially of the amino acid sequence of SEQ ID NO:
 3. 49. Apolypeptide comprising the amino acid sequence of SEQ ID NO: 3.